This is a renewal application for support for year 27 of Cell Transformation by Bovine Papillomavirus (R01 CA37157). This grant supported the discovery of the unique mechanism of action of the bovine papillomavirus E5 oncoprotein, a 44-amino acid very hydrophobic protein that is essentially an isolated transmembrane domain. We showed that the E5 protein binds to the transmembrane domain of the PDGF ? receptor, resulting in ligand-independent receptor activation. Detailed analysis of this interaction is providing new insights into transmembrane domain recognition and allowed us to develop a novel approach to manipulate cell behavior with artificial, small transmembrane proteins modeled on the viral protein. In an exciting advance in this funding period, we reprogrammed the E5 protein so that it no longer recognizes the PDGF receptor, but instead specifically activates the human erythropoietin receptor, driving erythroid differentiation of human hematopoietic progenitor cells. We propose here to extend our in-depth analysis of the interaction of the E5 protein and the PDGF ? receptor, with a particular attention to obtaining novel insights into the molecular basis of specific protein recognition within cell membranes, a process that underlies numerous biological processes, including many cancers. We will also develop small transmembrane proteins as potent biological agents against cancer and related diseases. We will isolate E5 mutants that recognize PDGF ? receptor mutants, to determine the rules responsible for highly-specific interactions between transmembrane helices. We will use related approaches to identify and characterize E5 mutants that inhibit PDGF ? receptor signaling. We will construct and analyze peptides to characterize the interaction between the E5 protein and the PDGF ? receptor transmembrane domain at the biochemical level. Finally, we will isolate and characterize small transmembrane proteins that modulate the HIV co-receptor, CCR5, a multipass transmembrane protein. These experiments should continue to provide important new insights into an unusual viral oncogene product and protein recognition within cell membranes, as well as providing new approaches to modulate important biological processes.